1. Field of the Invention
The present invention relates to the field of microscopic particulate devices made of biologic material suitable for intravenous administration, their methods of production and the use of such devices inside the body to treat medical conditions. More specifically, the present invention relates to the field of protein particles smaller than five micron in any one dimension, which are inert by themselves but serve as devices to capture or concentrate or carry biological, or drug molecules already in the blood, such that a combination of the device with captured biological or drug molecule can have unexpected or enhanced medicinal value.
One particular application is in the field of hemostasis, where after intravenous infusion, the device may capture molecules inside the body, such as a single component or a variety of coagulation factors, which then render the combination of device plus biological molecule capable of decreasing blood loss or shortening (improving) bleeding time. The mechanism of capture of endogenous molecules is unknown, nor is the exact mechanism of medicinal benefit.
These devices are expected to greatly benefit patients with insufficient platelet concentrations or diminished platelet function, or in patients with tendencies of bleeding due to other causes. Patients that are expected to benefit included thrombocytopenic patients from dilutional thrombocytopenia, cancer, thrombocytopenia from cancer treatments, idiopathic thrombocytopenia purpura, aplastic anemia, transplant patients, anticoagulant overdose, antiplatelet medication overdose and hemorrhagic episodes such as from Ebola or Dengue fever outbreaks. These devices can potentially be used for treatment as well as prophylactic use such as in surgical patients where perioperative blood loss is expected to be extensive but erythrocyte or platelet transfusion services are lacking; or in battlefield conditions where transfusion services are difficult.
2. Description of the Prior Art
Conventional methods of drug administration include oral, intravenous, intramuscular, subcutaneous, intraperitoneal, inhalation routes, nasal and mucosal applications. Numerous authors, including Yen had disclosed method of production and products where drugs can be encapsulated on the surface or interior of protein spheres for the purpose of targeted delivery to specific organs or sites to decrease systemic toxicity of the drug and/or enhance effectiveness at the site of action, particularly after intravenous infusion.
In the U.S. Pat. No. 5,069,936 (“Manufacturing Protein Microspheres”) Yen disclosed a method of producing albumin spheres by two methods (Column 4, line 53 to Column 5, line 28.) Both methods emphasized the importance of adding a surfactant or detergent to the protein solution before the formation of spheres from the protein solutions. One method (“Pre-link” method) used a cross-linking agent in a concentration sufficient to mildly cross-link protein molecules in a solution without the formation of a gel, before the addition of a desolubilizing or desolvation agent (such as ethanol) to form spheres. This concentration of crosslinking agent was, however, capable of binding the protein molecules together irreversibly in the form of spheres, even if the desolvation agent was subsequently diluted or removed. Another method (“Post-link” method) formed (reversible or resolubilizable) spheres first by the addition of a desolubilizating agent to a solution of protein. This step needs to be followed by the addition of a cross-linking agent in a concentration sufficient to stabilize the spheres against resolubilization during further processing (when the desolvation agent would be diluted or removed.)
U.S. Pat. No. 5,069,936 not only disclosed how to manufacture protein spheres but also taught that the spheres produced by either disclosed method can be used to bind other biological or drug molecules by covalent-bonding the drug to the spheres via a crosslinking agent (e.g. IgG, Example 7, Column 29) before administration to the patient. This was essentially achieved by adding an additional amount of crossing linking molecules (such as glutaraldehyde) after the spheres had been formed and irreversibly stabilized against resolubilization. This additional step of adding a crosslinking agent was intended to bind additional molecules to the spheres and not for the formation or stabilization of the spheres themselves. The biological activity of such spheres covalently bonded with biological or drug molecules (internally or on the surface of the spheres) entirely comes from the bound biological or drug molecules. In fact, control “blank” spheres (i.e. protein microspheres with no biomodifying agents, Example 9, Column 31) had been shown to demonstrate no efficacy as compared to a soluble drug such as adriamycin, or the same drug (adriamycin) encapsulated within the spheres.
The present invention described a series of novel methods to produce protein spheres without the need of adding or including surfactants or detergents to the protein solution before the formation of spheres. The sphere suspensions produced contained monodispersed spheres without aggregates and without large spheres (larger than 5 micron in diameter) that can clog capillaries when infused intravenously to patients.
Another advantage of the product made with the present invention is their small size. Because the spheres can be made with the present invention to be smaller than 1 micron in diameter, they tend to remain in suspension without sedimentation during prolong storage. Therefore, there is no need to lyophilize the suspension which was necessary with the preparations made with the disclosed methods in the prior art. Even with the expensive step of lyophilization with products made with the prior art, upon reconstitution of the lyophilized power with a fluid, the large spheres produced by the prior art can still sediment from the suspension and form a cake after some time.
Another property of the spheres manufactured with the series of novel methods disclosed in this invention is their efficacy in treating bleeding from thrombocytopenia. It is totally unexpected that upon certain changes in the method of production to be described in this application, that control or “blank” spheres, after intravenous infusion into thrombocytopenic animals, demonstrated hemostatic efficacy. These spheres did not have any fibrinogen or other coagulation (or other drugs) bound to them during the synthesis procedure. And spheres produced by this novel method after being kept at room temperature as a suspension for prolong periods of time were still efficacious.
The mechanism of shortening the bleeding time of thrombocytopenic animals by these novel “blank” spheres or microscopic devices made with the present invention is unknown. One can theoretically postulate a number of mechanisms, including the ability of these novel devices to bind biological molecules in vivo, such as any number of coagulation factors in any number of combinations. The potential binding of such molecules occurs after the infusion of such devices into the body. Since the exact identity and the concentration of biological molecules that hypothetically bind spontaneously to the spheres are difficult to ascertain and the molecules come from the blood in circulation and not added in vitro outside the body, such spheres essentially serve as devices. The devices apparently have the proper properties to capture, or concentrate, or carry the appropriate biological molecules to bring about the clinical efficacy. The device, however, without the appropriate additional molecules introduced in vivo is not expected to have by itself any medicinal efficacy.
It is further emphasized that the mechanism of this hypothetical combination of device with effective molecules (called “activated devices” from hereon) is also unknown, but can take the form of targeting the device to a wound site, or enhancement of endogenous platelet function (in a thrombocytopenic condition or in a patient with normal platelet concentration), or any number of unknown mechanisms.
In another patent, U.S. Pat. No. 6,264,988 B1 (“Fibrinogen-coated Microspheres”) Yen disclosed products and methods of production where fibrinogen molecules are attached to stabilized albumin spheres. Again, the spheres were made with added surfactant or detergent in the protein solution before the addition of a desolvation agent. However, the disclosed methods of production resulted in suspensions of spheres with a minority population of spheres which are too large in size. The patent described the use of filtration or centrifugation to remove “large particles” (larger than 7 micron in diameter, column 8, which will obstruct blood capillaries) before or after the addition of fibrinogen molecules to the spheres. The spheres produced by the present invention, by contrast, are all smaller than 5 micron and therefore the suspension requires no filtration or centrifugation steps. In particular, and of great relevance to the present invention is the disclosure in U.S. Pat. No. 6,264,988 B1 the lack of efficacy of “control spheres” (CS) which are albumin spheres without added fibrinogen, made by said disclosed methods. CS showed (as expected) no in vivo activity in the correction of bleeding time (FIG. 6) or the amount of blood loss (FIG. 7B) among thrombocytopenic rabbits. In vitro studies using ADP to aggregate mixtures of CS with human platelets showed non-participation of CS while human platelets form pure platelet aggregates (FIG. 14B.)
Therefore it is totally unexpected and novel that the presently disclosed new method of production of protein spheres without the addition in vitro of a biomodifying molecule such as fibrinogen or other clotting factor(s) can result in a product which has in vivo biological and medicinal efficacy.
Attempts to study the mechanism of such efficacy is expected not to be fruitful for several reasons: (1) It would be difficult to recover infused protein particles from the circulation compartment of the infused subject; which contains small particles such as platelet fragments, endothelial cell debris and other protein aggregates; (2) Any recovered particles are only a fraction of the infused population of particles. It is well known that rheologically smaller particles flow near the wall of capillaries and blood vessels, while larger particles flow near the centre of the blood vessels. Any studies performed on recovered particles only revealed the property of the recovered population of particles, probably collected from near the center of the circulatory compartment and not the original population of devices; (3) The biomodifying molecule is derived from the blood compartment in vivo which becomes attached to the device in vivo. It may detach from the particle after isolation, particularly in purification steps designed to remove soluble plasma proteins and other blood elements, e.g. red cells and platelets.
However, in vitro studies can offer some insights as to what these particulate devices can bind when mixed with plasma or fibrinogen solutions in vitro. These will be discussed as each experiment is described in the following sections.
In another patent (U.S. Pat. No. 5,725,804 “Non-Crosslinked Protein Particles for Therapeutic and Diagnostic Use”) Yen described a method of making spheres that will not redissolve upon removal or dilution of the desolvation agent. The mechanism of stabilization is unknown but can be achieved by the addition of a number of unrelated chemicals and drugs, all of which are not crosslinking agents. This prior art provides no motivation for anyone skilled in the art to use a crosslinking agents in the manner with which those non-crosslinking agents were used because the specific purpose of that patent was to teach how non-crosslinking agents could stabilize spheres without the presence or addition of crosslinking agents in any of the production steps. It is therefore unexpected and novel and non-obvious that the series of methods to be described here can produce sphere suspensions in the absence of added surfactants or detergent in the protein solution, and result with crosslinked spheres possessing desirable properties and medicinal efficacy.
In an abstract, published in the Journal of Thrombosis and Haemostasis, vol 5, supp 1, August, 2007, Appleby et al. described the design of a “platelet substitute” using human albumin microparticles to bind the fibrinogen-binding peptide Gly-Pro-Arg-Pro (GPRP). So, in stead of binding fibrinogen directly onto their microparticles, these investigators bound an intermediate peptide (GPRP) onto the microparticles. Their approach of binding fibrinogen indirectly onto their microparticles is obviously non-novel. Published data had shown that a link between a particle (such as a red cell) and a fragment from the fibrinogen molecule can facilitate the attachment of the particle (called “thromboerythrocyte”) to platelets. (see “Thromboerythrocytes” by Coller B S, et al, in J. Clin. Invest 1992, 89:546-555.) In contrast, data to be presented below for this invention showed that no intermediate molecules are needed on the spheres made with this invention, in order to bind fibrinogen onto the sphere either in vitro or in vivo.